Rat Adrenal Membranes Research Articles

Octarphin--Nonopioid Peptide of the Opioid Origin

The data on the properties and mechanism of action of the peptide octarphin (TPLVTLFK, the fragment 12-19 of β-endorphin)--a selective agonist of nonopioid (insensitive to the action of the opioid antagonist naloxone) β-endorphin receptor found on n immune cells (peritoneal macrophages, T and B lymphocytes of spleen and blood), endocrine (adrenal cortex, hypothalamus), cardiovascular (cardiomyocytes) systems are analyzed and systematized. Binding to the receptor octarphin increases increases the mitogen-induced pro- liferation of human and mouse T and B lymphocytes in vitro, activates murine peritoneal macrophages in vitro and in vivo, stimulates growth of human T-lymphoblast cell lines Jurkat and MT-4, inhibits adenylate cyclase activity of rat adrenal cortex membranes and suppresses the secretion of glucocorticoids from the adrenal gland into the blood. It was shown that in a concentration range of 1-1000 nM the peptide increases the activity of inducible NO-synthase (iNOS), and the content of NO and cGMP in lipopolysaccharide-activated murine peritoneal macrophages. Taking into account that NO acts as a primary activator of soluble guanylate cyclase (sGC), it can be assumed that the activating effect of octarphin on macrophages is realized in the following way: increase in th iNOS expression --> increase in the NO production --> increase in the sGC activity --> increase in intracellular levels of cGMP.

3H]Metyrapol and 4-[131I]Iodometomidate Label Overlapping, but Not Identical, Binding Sites on Rat Adrenal Membranes

Metyrapone, metyrapol, and etomidate are competitive inhibitors of 11-deoxycorticosterone hydroxylation by 11β-hydroxylase. [(3)H]Metyrapol and 4-[(131)I]iodometomidate bind with high affinity to membranes prepared from bovine and rat adrenals. Here we report inhibitory potencies of several compounds structurally related to one or both of these adrenostatic drugs, against the binding of both radioligands to rat adrenal membranes. While derivatives of etomidate inhibited the binding of both radioligands with similar potencies, derivatives of metyrapone inhibited the binding of 4-[(131)I]iodometomidate about 10 times weaker than the binding of [(3)H]metyrapol. By X-ray structure analysis the absolute configuration of (+)-1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanol [(+)-11, a derivative of metyrapol] was established as (R). We introduce 1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanone (9; Ki = 6 nM), 2-(1-imidazolyl)-2-methyl-1-phenyl-1-propanone (13; 2 nM), and (R)-(+)-[1-(4-iodophenyl)ethyl]-1H-imidazole (34; 4 nM) as new high affinity ligands for the metyrapol binding site on 11β-hydroxylase and discuss our results in relation to a proposed active site model of 11β-hydroxylase.

Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes

The synthetic peptide octarphin (TPLVTLFK, fragment 12-19 of β-endorphin), a selective agonist of the non-opioid β-endorphin receptor, was labeled with tritium yielding specific activity of 28 Ci/mmol. The binding of [3H]octarphin to rat adrenal cortex membranes was studied under normal conditions as well as after cold and heat shocks. It was found that under normal conditions [(3)H]octarphin specifically binds to the membranes with high affinity: K(d1) = 36.3 ± 2.5 nM, B(max1) = 41.0 ± 3.8 pmol/mg protein. The specific binding of [(3)H]octarphin to the membranes was inhibited by unlabeled β-endorphin (K(i) = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (K(i) = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu(5)]- and [Met(5)]enkephalins, α- and γ-endorphins, and corticotropin were inactive (K(i) > 1 µM). Both cold and heat shocks decreased the binding affinity: K(d2) = 55.6 ± 4.2 nM and K(d3) = 122.7 ± 5.6 nM, respectively. In both cases, the maximal binding capacity of the receptor did not change. Thus, even a short-term thermal shock significantly affects the sensitivity of the non-opioid β-endorphin receptor of adrenal cortex membranes.

Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors

Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX-binding sites. Specific [125I]WTX binding was partially inhibited by both alpha-cobratoxin, a blocker of the alpha7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0+/-0.8 microM and was inhibited by alpha-cobratoxin but not by a short-chain alpha-neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the alpha7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 microM did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.

New Selective Inhibitors of Steroid 11β-Hydroxylation in the Adrenal Cortex. Synthesis and Structure–Activity Relationship of Potent Etomidate Analogues

Derivatives of etomidate were evaluated as inhibitors of adrenal steroid 11beta-hydroxylations. Stereoselective coupling by Mitsunobu produced chirally pure analogues to study the effect of configuration, modification of the ester, and substitution in the phenyl ring, with the aim to probe specific sites for introducing a radionuclide. Iodophenyl metomidate (IMTO) labeled with iodine-131 served as radioligand for structure-affinity relationship studies. We have characterized the kinetic parameters of specific (131)I-IMTO binding on rat adrenal membranes and used the displacement of (131)I-IMTO binding to evaluate functionalized MTO analogues. Our results indicated that (1) ( R)-configuration is essential for high affinity, (2) highest potency resides in the ethyl, 2-propyl, and 2-fluoroethyl esters, and (3) substitution of the phenyl ring is well tolerated. The clinically used inhibitors metyrapone and ketoconazole inhibited (131)I-IMTO binding with low affinity. Incubation of selected analogues with human adrenocortical NCI-h295 cells demonstrated a high correlation with the inhibitory effect on cortisol secretion.

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.

Elucidation and characteristics of non-opioid beta-endorphin receptors in rat adrenal cortex.

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 +/- 2.0 nM, Bmax1 = 40.7 +/- 2.3 pmol/mg protein; Kd2 = 0.25 +/- 0.01 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10(-9)-10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Characteristics of non-opioid beta-endorphin receptor.

Tritium-labeled selective agonist of non-opioid beta-endorphin receptor, the decapeptide immunorphine ([3H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid beta-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [3H]immunorphine-receptor complex was 2.4 +/- 0.1 nM) and beta-endorphin (Ki of the [3H]immunorphine specific binding was 2.9 +/- 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro3]pentarphine (VKPFY) (Ki values were 0.0060 +/- 0.0004, 2.7 +/- 0.2, 2.6 +/- 0.2, and 2.8 +/- 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 microM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [3H]immunorphine. Values of the specific binding of 8.4 nM [3H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 +/- 44.7, 698.6 +/- 28.1, 279.1 +/- 15.4, and 172.2 +/- 1.8 fmol/mg protein, respectively. Unlabeled beta-endorphin, pentarphine, [Pro3]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [3H]immunorphine to membranes from these organs. No specific binding of [3H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

123/131I]Iodometomidate as a radioligand for functional diagnosis of adrenal disease: synthesis, structural requirements and biodistribution

Abstract Metomidate [(R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid methyl ester] (MTO, 1, Fig. 1) is a potent and selective inhibitor of the cytochrome P-450 enzyme system in the adrenal cortex. Labelled in the 4-position with radioiodine, (R)-4-[131I]iodometomidate, 2, [131I]IMTO has been evaluated by in-vitro studies and also ex-vivo in rats. [131I]IMTO was synthesized by oxidative radioiododestannylation using a suitable precursor which was prepared by a new stereoselective synthesis. Optimization of the labelling reaction was performed by systematic variation of the most important reaction parameters. Under optimum reaction conditions, a labelling yield of 95% was obtained. In-vitro-stability of the tracer was studied over 8 days, indicating slow deiodination (0.27%/h). Displacement studies using [131I]IMTO and rat adrenal membranes revealed the structural requirements for high affinity binding, namely an intact ester group and (R)-configuration of the radioligand. Pharmacokinetic studies in rats showed fast accumulation of [131I]IMTO in the adrenals (approx. 10% ID/g tissue) with an activity plateau for 2 hours. Metabolic degradation was indicated by a steady increase of renal activity up to 4 hours post injection. Based on target to non-target ratios the highest contrast for imaging of the adrenals was observed between 30 and 60 min post injection of [131I]IMTO. We conclude that SPECT using [123I]IMTO will be a promising method for the characterization of adrenal incidentalomas.

In vitro pharmacology of an angiotensin AT 1 receptor antagonist with balanced affinity for AT 2 receptors

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2′-( N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1′]-biphenyl-4-yl]methyl]-3 H-imidazo[4,5- b]pyridine) inhibited specific 125I-[Sar 1,Ile 8]angiotensin II binding to angiotensin AT 1 receptor ( K i = 0.11−0.20 nM) in rabbit aorta, rat adrenal and human angiotensin AT 1 receptor in CHO (Chinese hamster ovary transformed) cells and to AT 2 receptor ( K i = 0.14−0.23 nM) in rat adrenal and brain receptors. L-163,017 also had a high affinity in the presence of bovine serum albumin (2 mg/ml), for angiotensin AT 1 and AT 2 receptors on human adrenal ( K i 3.9 and 4.3 nM), aorta ( K i 0.45 and 0.96 nM) and kidney ( K i 3.6 and 2.3 nM). The much higher K i values in human tissues were likely due to the presence of bovine serum albumin in the binding assay buffer since L-163,017 had K i values of 0.13 ± 0.04 and 2.0 ± 0.04 nM in the absence and presence of bovine serum albumin, respectively, in inhibiting 125I-[Sar 1,Ile 8]angiotensin II binding to angiotensin AT 1 receptor in rat adrenal membranes. Scatchard analysis of 125I-[Sar 1,Ile 8]angiotensin II binding in the presence of bovine serum albumin (2 mg/ml) in rabbit aorta and bovine cerebellum indicated a competitive interaction of L-163,017 with angiotensin AT 1 and AT 2 receptors ( K i values 2.5 and 2.1 nM respectively). L-163,017 inhibited angiotensin II-induced aldosterone release in rat adrenal demonstrating that L-163,017 acted as a competitive antagonist (pA 2 = 9.9) and lacked agonist activity. L-163,017 also inhibited angiotensin II responses in rat vascular tissues. The specificity of L-163,017 was shown by its lack of activity on the above functional responses produced by other agonists and in several binding assays.

Pharmacology of XR510, a Potent Orally Active Nonpeptide Angiotensin II AT1 Receptor Antagonist with High Affinity for the AT2 Receptor Subtype

The angiotensin II (Ang II) type 1 receptor (AT1) mediates all known physiological effects of ANG II, whereas functions of the type 2 (AT2) receptor are not clear. Should undesirable AT2 effects be identified, it may be advantageous to combine antagonism of AT1 and AT2 receptors. XR510 was shown to inhibit the specific binding of [125I]Sar1,Ile8-Ang II for AT1 and AT2 subtype binding sites in rat adrenal membranes with IC50 of 0.26 and 0.28 nM, respectively, and in human tissues with subnanomolar binding affinity. In isolated rabbit aorta, XR510 exerted insurmountable Ang II antagonism with a Kb value of 4 nM. In conscious renal hypertensive rats, XR510 decreased blood pressure (BP) with intravenous (i.v.) and oral (p.o.) ED30 of 0.08 and 0.27 mg/kg, respectively. In spontaneously hypertensive rats (SHR), repeated daily oral dosing of XR510, losartan, and enalapril at 30 mg/kg/day decreased BP similarly. In conscious furosemide-treated dogs, XR510, given either intravenously or orally, decreased BP. These results suggest that XR510 is an orally active and selective Ang II receptor antagonist with equal binding affinities for AT1 and AT2 receptor binding sites.

In vitro pharmacological characterization of UP 269-6, a novel nonpeptide angiotensin II receptor antagonist.

f1p4in vitro pharmacology of UP 269-6, a novel nonpeptide angiotensin II antagonist, was examined in radioligand binding and functional isolated tissue assays. UP 269-6 bound selectively to AT1 receptors as evidenced by the inhibition of specific [125I] Sar1, Ile8-AII binding in rat adrenal membranes (IC50 = 35.8 nM) and in cultured vascular smooth muscle cells (IC50 = 23.8 nM). UP 269-6 displayed a very high selectivity for the AT1 compared to the AT2 receptor subtype (IC50 > 10,000 nM). UP 269-6 inhibited the AII-induced contraction of isolated rabbit aortic strips. The pattern of AII antagonism suggested competitive antagonism at low concentrations (10(-10), 3 x 10(-10), 10(-9) M) of UP 269-6 and insurmountable antagonism at higher concentrations (3 x 10(-9), 10(-8), 3 x 10(-8) M). Based on the calculated pA2 values, UP 269-6 (9.86 +/- 0.25) was an angiotensin II receptor antagonist as potent as L-158,809 (9.82 +/- 0.37) and much more potent than losartan (7.96 +/- 0.38). UP 269-6 was devoid of affinity (IC50 > 10,000 nM) for many other receptors, ion channels and uptake sites, demonstrating its high specificity for AII receptors. Furthermore, this compound did not affect the contractile response to KCl or phenylephrine in rabbit aorta and exhibited no effect on angiotensin converting enzyme activity. These data demonstrate that UP 269-6 is a highly potent, selective and specific AT1 receptor antagonist.

2-(125I) iodomelatonin binding sites in rat adrenals: Pharmacological characteristics and subcellular distribution

Specific binding sites for 2-[125I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[125I] iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin > 2-iodomelatonin > melatonin > 5-methoxytryptamine > 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear (0.76 fmol/mg protein) and mitochondrial (1.82 fmol/mg protein) subcellular fractions.

Regulation of particulate guanylate cyclase by atriopeptins: relation between peptide structure, receptor binding, and enzyme kinetics

Structural analogs of atriopeptins (APs) were compared for their ability to activate particulate guanylate cyclase and bind to specific receptors in rat adrenal membranes. All analogs tested increase V max without altering the concentration of substrate required for half-maximum activity or the positive coperativity exhibited by the enzyme. Maximum velocities (pmoles of cGMP produced per min per mg protein) achieved in the absence and presence of APs were 128.3±6.6 and 283.8±20.6 using Mn 2+-GTP, and 53.7±3.7 and 149.9±7.6 using Mg 2+-GTP as the substrate, respectively. Although all APs were equally efficacious in activating the enzyme, their rank potency was ANF (8–33) = AP III = AP II > AP I when either divalent cation was used as the cofactor. The EC 50 for activation of guanylate cyclase by AP I was about 10 −7 M, while that for the other peptides was about 10 −8 M, using either divalent cation cofactor. 125I-labeled ANF bound to rat adrenal membranes with a K D of 5·10 −10 M. Although all APs were equally efficacious in competing with labeled ANF for receptor binding, their rank potency was identical to that for enzyme activation. The K i for AP I was about 10 −8 M, while that for the other peptides was about 10 −10 M. These data suggest that the carboxy terminal Phe-Arg present in the AP analogs except AP I and critical for biological and receptor-binding activity are also important in coupling receptor-ligand interaction with guanylate cyclase activation. The correlation between the rank order potency for receptor binding, enzyme activation, and the reported physiological actions of APs support the suggestion of a functional coupling between these proteins.

Receptors for insulin and insulin-like growth factor-I in the human adrenal gland

Human adrenal glands contain high-affinity receptors for insulin and insulin-like growth factor I (IGF-I). Comparative studies with rat, hamster and human adrenal membranes confirmed that IGF-I receptors are most abundant in rat and hamster adrenals, whereas insulin and IGF-I receptors are present in equivalent numbers in human adrenal glands. Covalent crosslinking studies revealed that the human adrenal gland IGF-I receptor binding subunit migrated on dodecyl sulfate polyacrylamide gels with Mr = 135,000, which is identical to the migration of IGF-I receptor binding subunits isolated from other tissues. Autoradiography of frozen human adrenal slices incubated with [ 125I]insulin showed prominent, displaceable binding of this radioligand to the zona reticularis, zona glomerulosa, vasculature and medulla; in contrast, [ 125I]IGF-I binding to human adrenal tissue was most prominent in the zona reticularis and negligible in the medullary region.

The use of complementary peptides in the purification of an angiotensin II binding protein.

We used the molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, to purify an angiotensin II (Ang II) binding protein. The complementary peptide IIA (Lys-Gly-Val-Asp-Val-Tyr-Ala-Val) specified by the RNA sequence complementary to the messenger (m)RNA sequence for rat Ang II was synthesized, purified and used to raise polyclonal antibodies. Complementary peptide IIA specifically inhibited the binding of 125I-Ang II to receptors on rat adrenal membranes, and anti-IIA immunoglobulin G (IgG) specifically inhibited the binding of 125I-Ang II to rat adrenal Ang II receptors and Ang II-dependent aldosterone secretion by cultured rat adrenal cells, suggesting that the antibody recognizes the Ang II receptor. Anti-IIA IgG was used for immuno-affinity purification, from a rat adrenal membrane preparation of an Ang II binding protein with a molecular weight of 66,000 +/- 2000 that bound 125I-Ang II specifically. This is the first report of purification of an Ang II receptor binding protein which retains its capacity to specifically bind 125I-Ang II.

Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA.

We have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. We demonstrate that the antibody competes specifically with 125I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, we show that this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, we demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of Mr 66,000 that specifically binds radiolabeled AII.

The effects of vanadium compounds on the activation of adenylate cyclase from rat adrenal membrane

In rat adrenal membrane, vanadyl sulfate, but not vanadate, inhibits the nonhydrolyzable GTP analogs-,forskolin- and NaF-stimulated activation process of adenylate cyclase. In these reactions, the half-maximum concentration of vanadyl for inhibition was approx. 0.3 mM. The binding of [ 3H]guanyl-5′-yl imidodiphosphate to the membrane ( K d = 2 μ M ) was not affected by vanadyl sulfate under the conditions in which the vanadyl sulfate inhibits the activation process. Also, the binding of ACTH to its receptor was inhibited by neither vanadyl sulfate nor vanadate, and the catalytic unit of adenylate cyclase appears to be unaffected by vanadyl sulfate. When the activation by nonhydrolyzable GTP analog was enhanced by Ca 2+, vanadyl sulfate strongly inhibited the activation of adenylate cyclase.

Inhibition by vanadyl of adenylate cyclase activation reactions in rat adrenal membranes

In rat adrenal dispersed cells, both vanadyl and vanadate inhibited ACTH-stimulated steroidogenesis and the formation of cAMP, whereas these compounds did not inhibit the cAMP-dependent steroidogenesis. Then, the membrane fraction was prepared and activated by various secretagogues including ACTH, Gpp(NH)p, GTPγS, and forskolin. The cyclase activity was inhibited by vanadyl but not by vanadate in the presence of these stimulators. Based on these results, we conclude that cationic vanadyl acts against a metal-requiring step in the adenylate cyclase system containing G-protein and the catalytic subunit. In addition, we believe that vanadyl is a useful tool to investigate adenylate cyclase systems.

Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system.

Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of [125I]rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.